Download Production and identification of haploid dwarf male sterile wheat

Zhang et al. Botanical Studies 2014, 55:26
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RESEARCH
Open Access
Production and identification of haploid dwarf
male sterile wheat plants induced by corn
inducer
Wei Zhang†, Ke Wang†, Zhi-Shan Lin†, Li-Pu Du, Hai-Li Ma, Le-Le Xiao and Xing-Guo Ye*
Abstract
Background: Using the cross of wheat and maize is a very useful way to produce wheat haploid plants by
chromosome elimination. Dwarf male sterile wheat (DMSW) and corn inducer are potential important germplasm
for wheat breeding by recurrent selection and doubled haploid strategies. There is no report yet to achieve the
haploid plants from DMSW induced by maize inbred line and especially the corn inducer.
Results: Haploid plants of DMSW were successfully obtained in this study induced by both maize pollens of
inducer line and normal inbred line. The efficiencies for wheat embryos formation and plantlets production induced
by the two corn lines had no significant difference. All the eleven haploid wheat plants derived from the male
sterile material were identified by botanic appearance, cytology, cytogenetics, and molecular markers. They were all
haploid based on their guard cell length of 42.78–42.90 μm compared with the diploid control of 71.52 μm, and
their chromosome number of 21 compared with the diploid control of 42. In addition, according to anthers, plant
height, and molecular markers, the haploid plants were divided into two types. Eight of them showed dwarf,
having no anthers, and the special band of Rht10, and the other three plants displayed normal plant height, having
anthers, and not containing the special band of Rht10, indicating that they were originated from the MS2/Rht10
and ms2/rht10 female gametes, respectively.
Conclusions: MS2/Rht10 haploid plants were successfully obtained in this study by using corn inducer and inbred
line, and will be employed as candidate materials for the potential cloning of MS2 dominant male gene.
Keywords: Corn inducer; Dwarf male sterile wheat; Intergeneric cross; Ms2 haploid plant
Background
Wheat is an economic valuable crop species worldwide,
and its continual genetic improvement on yield, disease
resistance, processing quality, and abiotic stress tolerance
is closely associated with food security production for
human being (Shewry 2009; Tester and Langridge 2010;
He et al. 2011). In wheat breeding program germplasm
takes an essential role for the development of new varieties. Wheat germplasm with various distinguished
characteristics can be identified in natural population
or bred by commercial and wild cross and mutation
* Correspondence: [email protected]
†
Equal contributors
National Key Facility for Crop Gene Resources and Genetic Improvement, Key
Laboratory of Biology and Genetic Improvement of Triticeae Crops of
Ministry of Agriculture, Institute of Crop Sciences, Chinese Academy of
Agricultural Sciences, Beijing 100081, China
(He et al. 2011; von Well and Ncala 2013). Of them,
male sterile materials are beneficial to hybrid applications
and recurrent selection of wheat (Zhai and Liu 2009). Up
to date, three key male sterile genes have been found in
wheat. A recessive male sterile gene, ms1, was found to be
located on chromosome 4A (Driscooll 1978), but this gene
was not convenient to be employed in wheat breeding
procedure. A dominant male sterile gene, MS3, was obtained from the nuclear-cytoplasm hybrid between wheat
and Aegilops squarrosa after treatment by ethyl methane
sulfonate (EMS) (Maan et al. 1987; Qi and Gill 2001).
An important wheat male sterile mutant was found in
1972 by Gao (1987). Genetic analysis demonstrated that
its male sterility was controlled by a single dominant gene
Tal (Deng and Gao 1980, 1982), which was renamed as
Ms2 later (Cao et al. 2009; Zhai and Liu 2009). Ms2 exists
in wheat plants always in heterozygous status of Ms2ms2,
© 2014 Zhang et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly credited.
Zhang et al. Botanical Studies 2014, 55:26
http://www.as-botanicalstudies.com/content/55/1/26
and inherits itself by accepting the pollens of normal wheat
varieties with ms2ms2 genotype at this locus (Deng and
Gao 1980, 1982, 1987; Gao 1987). Ms2ms2 plants have no
anthers completely while their pistils are developed normally, and their next generation always produced sterile
plants by half with genotype Ms2ms2 and fertile plants by
another half with genotype ms2ms2 (Deng and Gao 1980,
1982, 1987; Zhai and Liu 2009). By using telosomic mapping and genetic analysis approaches, this gene was located
on the short arm of chromosome 4D, and 31.16 cM away
from the centromere (Liu and Deng 1986a, b). To identify
the Ms2 gene and distinguish male sterile plants in early
stage, a dwarf male sterile wheat (DMSW) was further developed by crossing the male sterile Chinese Spring with
Aibian 1 carrying the dwarfing gene Rht10 (Rht-D1c) and
backcrossing the dwarfing F1 male sterile plants with
normal Chinese Spring, in which Ms2 gene was linked
closely with Rht10 gene with a genetic distance of 0.12 cM
(Liu and Yang 1991; Zhai and Liu 2009). Since then, the
dwarf male sterile wheat has been efficiently applied in
wheat breeding for population improvement through recurrent selection and genetic variation expansion through
easily cross-making (Liu et al. 2002), and a number of new
wheat varieties have been developed by using the special
material (Zhai and Liu 2009). However, Ms2 has not been
successful cloned and a perfect direct molecular marker
for it has not been developed yet. The main problem is that
the male sterile gene always exists in heterozygous state
and the DNA samples are mixed with ms2 from male gametes in this kind of dwarf male sterile wheat plants. Therefore, it is necessary to obtain haploid or double haploid
male sterile plants derived from the Ms2 female gametes.
Cao et al. (2009) found that WMC617 marker was closely
linked to the male sterile Ms2 gene and the dwarfing Rht10
gene among 48 pairs of SSR primers by bulked segregation
analysis, and further mapped the two genes at the distal
position of chromosome 4DS. Li et al. (2012) revealed
that the Rht10 allele contained in DMSW was generated
through a tandem segmental duplication (TSD) of a region
more than 1 Mb carrying Rht2 (Rht-D1b) gene, resulting in
two copies of Rht2, namely that Rht10 was completely
identical to Rht2 in sequence. Further, they analyzed the
segregation population containing Rht10 gene by using
Rht2 gene-specific primers. It is implied that Rht10 in
DMSW also can be detected by the perfect PCR-based
marker pairs of DF-MR2 developed by Ellis et al. (2002).
Wild cross of wheat and maize is the ideal way to
induce female gametes originating wheat haploid plants
(Laurie and Bennett 1988a). This technique has now been
widely used in the production of wheat haploid plants
(Laurie and Reymondies 1991; Sun et al. 1992; Chen et al.
1996a, b). Sun et al. (1999) firstly achieved the Ms2 haploid
plants by crossing the male sterile wheat with normal inbred maize line, and identified the haploid by cytogenetics.
Page 2 of 8
Current study is trying to obtain Ms2/Rht10 haploids by
using the dwarf male sterile wheat and corn inducer, and to
compare the induction efficiency of corn inducer and normal inbred corn line. The obtained haploids will be identified by botanical traits, cytology, and molecular marker.
Results generated in this study will be useful for marker
development and isolation of Ms2 gene, and for the production of wheat haploid plants using corn inducer.
Methods
Plant materials
Dwarf male sterile wheat (DMSW) line DS987 was kindly
provided by Prof. Binghua Liu at the Institute of Crop
Sciences (ICS), Chinese Academy of Agricultural Sciences
(CAAS). Chinese Spring (CS) was demanded from the
National Crop Germplasm Preservation Center at the ICS,
CAAS. The pollens of corn inducer CI011-1 and Zheng58
were collected from the greenhouse of National Key Facility
for Crop Gene Resources and Genetic Improvement.
CI011-1 was developed by Biotechnology Research Institute
of CAAS using a corn inducer, HZI1, as one parent and
frequently employed for the production of maize haploid
plants by the scientists in our institute (ICS, CAAS).
Zheng58 was developed by Henan Xingyang Feilong Seed
Limited Company of China and normally used for the
induction of wheat haploid plants at ICS, CAAS.
Crossing and hybrid embryos rescue of DMSW and
corn inducer
The seeds of DMSW line DS987 were planted in the
greenhouse with a photoperiod of 14 h light/10 h darkness and a temperature regime of 25°C day/15°C night.
Once the heads of the male sterile plants were just
completed out of the sheaths of the flag leaves, the
spikes were prepared by removing the up and down
spikelets and the middle florets of each remaining
spikelet, and cutting the top part of the lemmas of each
floret with scissors. Then, the prepared spikes were
bagged with sulfuric acid paper. Three days later, the
bagged spikes were pollinated with the pollens of the
two maize lines by pouring from the top of the bags
with hand shaking. On the next 2 days, the pollinated
spikes were treated with 2,4-dichlorophenoxyacetic
acid (2,4-D) 100 mg L-1 containing solution for 2 times
(Chen et al. 1996a; Chen et al. 1996b). 14–15 days post
pollination, the immature grains were collected and
sterilized with 70% ethanol for 1 min, immersed in 20%
bleach for 15 min and rinsed with sterile distilled water
for five times inside laminar flow bench. The immature
embryos were isolated with inoculating needle under
anatomical lens and cultured in axial side up on germination medium (pH 6.0) containing 1/2 Murashige and
Skoog (1962) (MS) basal salts, 2.0% sucrose, B5 vitamins,
and 2.4 g L-1 phytagel at conditions of 25 ± 1°C, 16 h
Zhang et al. Botanical Studies 2014, 55:26
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photoperiod, 400 μmol m-2 s-1 photosynthetic photon
flux density and 45% relative humidity for 4 wk to obtain healthy haploid plantlets (Chen et al. 1996b,
2013). Finally, the plantlets were transplanted in soil in
natural environment condition for further investigation.
Test of percent hypothesis was used to evaluate the difference of corn inducer and normal inbred lines on inducing wheat haploid plants.
water until the thin achromatic epicuticula left. The stoma
guard cells on the epidermis were observed and measured
under an optical microscope incorporated with daily mirror
micrometer (Wang et al. 1989; Du et al. 1996). Each
plant was measured by ten guard cells for statistics, and
the data was statistically analyzed by SPPS13.0 Software
(SPSS Chicaco, USA) for significant difference and standard
deviation.
Root tips preparation and chromosome observation
Results
Cytological analyses followed the procedures described
by Lin et al. (2002). Roots excised from the rescue plantlets
and the DMSW control plants were treated in ice-water for
about 24 h, fixed in ethyl alcohol and acetic acid mixture
(3 : 1) for 24 h, and then transferred to 95% ethyl alcohol
and stored at −20°C until use. The fixed materials were hydrolysed in 1 N HCl at 60°C for 14 min, and then stained
with Schiff's reagent. Squashes of the relevant tissues were
made in a drop of 1% acetocarmine on slides. Chromosome
number was counted with microscope.
Obtaining of haploid dwarf male sterile wheat plants
assisted with the pollen of corn inducer
DNA extraction and PCR amplification
Wheat leaves were collected from the plants of generated
haploids between DMSW and corn inducer, DMSW
DS987, and CS at booting stage, and their genomic DNA
were extracted using modified cetyltrimethylammonium
bromide (CTAB) method [3% (w/v) CTAB, 1.4 M NaCl,
20 mM ethylenediaminetetraacetic acid (EDTA), 0.1 M Tris
HCl, pH 8.0, 2% (w/v) polyvinylpolypyrrolidone and 0.2%
(v/v) 2-mercaptoethanol] (Mahanil et al. 2007). The DNA
pellet was dissolved in sterile water.
A pair of PCR primers specific to Rht2 gene (Accession
number in Genbank: JF930281) of 5′-CGCGCAATTATT
GGCCAGAGATAG-3′ and 5′-CCCCATGGCCATCTCG
AGCTGCTA-3′ named as DF-MR2 (Ellis et al. 2002) were
used to identify whether the freshly produced haploid
wheat plants between DMSW and corn inducer contain
Rht2 gene or Ms2 gene. PCR reaction was performed in
a final volume of 20 μL consisting of 100 ng of genomic
DNA, 10 uL of 2 × Taq MasterMix (CwBio, China), 3 μM
of each primer and 7.4 μL of ddH2O. Amplification process
consists of 94°C for 5 min, seven “touchdown” cycles of
94°C for 30 s, 65°C for 30 s, 72°C for 1 min 20 s with a 1°C
drop in annealing temperature at each cycle, followed by
34 cycles of 94°C for 15 s, 58°C for 15 s, 72°C for 50 s, and
one cycle of 72°C for 10 min. PCR products were separated
on 2.0% agarose gel, and observed under UV light.
The prepared and bagged spikes of dwarf male sterile
wheat line DS987 three days ago were pollinated with
freshly collected pollens of corn inducer and normal
inbred line. After pollination and 2,4-D treatment for
15 days, in total twenty-eight and eleven developing
immature embryos were obtained from the induction
of corn inducer and normal inbred line, respectively,
and inoculated on the rescue medium for further growing.
Finally, eight healthy plantlets were initiated by the corn
inducer, and three by the normal inbred line (Figure 1).
Results suggested that the frequencies for the embryos
formation and plantlets induction induced by the corn
inducer line (5.00% and 1.43%, respectively) were slight
higher than those by the normal inbred line (3.44% and
0.94%, respectively), but the induction rate difference
between the two maize lines for wheat embryos and
Determination of the length of guard cells on wheat leaves
Leaf pieces of 2 cm in length were collected from each
plant at jointing stage, and put on the center of a glass
slide upward of the leaf abaxial epidermis. The samples
were fixed with left forefinger and the mesophyll tissues
were scraped off carefully by a scalpel with a little bit
Figure 1 The generated plants from the dwarf male sterile
wheat induced by corn inducer and normal inbred line. A: The
plantlet was induced by corn inducer; B: The plantlet was induced
by normal inbred corn line.
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Table 1 Production of haploid dwarf male sterile wheat plants induced by corn inducer and normal inbred line
Donors of
maize pollen
Florets
pollinated
Embryos
cultured
Plantlets
obtained
Embryos formation
rate* (%)
Plantlets induction
rate* (%)
CI011-1
560
28
8
5.00
1.43
Zheng58
320
11
3
3.44
0.94
*The embryo formation rate was calculated through the number of cultured embryos divided by the number of pollinated florets. The plantlet induction rate was
calculated via the number of obtained plants divided by the number of pollinated florets. The rates difference between the two maize lines for wheat embryos
formation and plantlets induction were not significant (P>0.05) by percent hypothesis test.
plantlets was not significant (P>0.05) according to percent
hypothesis test (Table 1).
Identifying of the haploid dwarf male sterile wheat plants
by cytology and botanical appearance
The obtained eleven plants from the wild cross were
examined for chromosome constitution before transplanted
into soil. Twenty-one chromosomes were observed in all
the rescued plants (Figure 2A), while the number was
forty-two in the plants of dwarf male sterile wheat line
DS987 (Figure 2B). Normally, the length of stoma guard
cells on abaxial epidermis of leaves is used to primarily
identify the ploidy of the plants from another culture.
The size of 61.0–63.0 μm was regarded to the boundary
of haploid and diploid wheat plants (Wang et al. 1989;
Du et al. 1996). Thereby, the generated plants were
measured for the length of guard cells on leaves at
jointing stage. The average length of the guard cells was
42.90 ± 2.84 μm (varied from 40.80 μm to 46.80 μm) and
42.78 ± 1.83 μm (varied from 40.80 μm to 45.10 μm) for
the short and high rescued plants, respectively (Figure 3A),
which were not different significantly between the cells and
plants. While, the corresponding value was 71.52 ± 4.62 μm
(varied from 67.20 μm to 79.20 μm) for DS987 plants
(Figure 3B). Above results demonstrated that all the generated plants from the distant cross were in haploid status.
Among the eleven haploid plants, three of them were about
75 cm in plant height (Figure 4A) and had three anthers in
each floret (Figure 5A), appearing normal plant height and
fertile botanical traits. And the other eight haploid plants
were 30 cm around in plant height (Figure 4B) and had no
anthers in the florets (Figure 5B), showing dwarfing and
male sterile characteristics. We believe that the haploid
plants with normal height and anthers were developed
from ms2/rht10 female gametes, and the plants showing
dwarfing and no anthers were originated from MS2/Rht10
female gametes. Once induced by maize pollens, the two
types of female gametes all can develop into embryos.
Identifying of the haploid dwarf male sterile wheat plants
by molecular marker
Ellis et al. (2002) developed a perfect PCR-based marker
pairs DF-MR2 to detect Rht2 gene. Therefore, all the generated haploid plants obtained in this study were detected
by the molecular marker for Rht10 and Ms2 genes. Eight
of the eleven plants amplified the specific band to Rht10
or Ms2, and the other three plants didn’t amplify the
specific band (Figure 6). The identification results of the
haploid plants by botanical traits and molecular markers
were matched in with each other, further confirming
that the two groups of plants were originated from the
MS2/Rht10 and ms2/rht10 female gametes, respectively.
Discussion
In the intergeneric cross between wheat and maize,
genotypes of the both generas exert some influences on
Figure 2 Chromosome constitution of the rescued plants derived from the dwarf male sterile wheat induced by maize pollens and
control diploid plants. A: The rescued haploid plants contained 21 chromosomes; B: DS987 control plants contained 42 chromosomes.
Zhang et al. Botanical Studies 2014, 55:26
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Figure 3 Leaf guard cells of the rescued plants derived from the dwarf male sterile wheat induced by maize pollens and control
diploid plants. A: The rescued haploid plants had smaller guard cells; B: DS987 plants had larger guard cells.
Figure 4 Height appearance of the haploid plants derived from
the dwarf male sterile wheat induced by maize pollens. A: The
ms2 haploid sterile plant showed normal height; B: The Ms2 haploid
sterile plant showed short height.
the formation of wheat haploid embryos. Using the same
maize pollens to be pollinated, different wheat lines all
could produce haploid embryos with various efficiencies
(Inagaki and Tahir 1990; Cai et al. 2004). For example,
Chen et al. (1998) reported the embryos formed rate was
ranging from 18.5% to 29.1%. The efficiencies were not
only related to the growth habits of wheat varieties, but
also to their origins (Laurie and Bennett 1988a; Suenaga
and Nakajima 1989; Inagaki and Tahir 1990) and environmental conditions (Chen et al. 2013). However, it was
demonstrated that the Kr gene in wheat background
had no effect on the crossability of wheat and maize
(Laurie and Bennett 1988b). On the other hand, a wheat
line also could generate haploid embryos when pollinated
with different maize genotypes, and sweet corn was better
than waxy corn and normal corn in the induction efficiency of wheat haploid embryos (Suenaga and Nakajima
1989; Wang 1998; Cai et al. 2004).
Coe (1959) identified a corn inducer, Stock6, that induced parthenogernesis of maize by a higher frequency.
Since then, a lot of improved corn inducers have been
developed using Stock6 as parent by hybrid technique, such
as WS14, Krasnodar Markers, MH1, M741H, ZMS, HZI1
(Lashermes and Beckert 1988; Chalyk 1994; Shatskaya et al.
1994; Eder and Chalyk 2002; Zhang et al. 2008; Elizabeth
and William 2009). Through the use of corn inducer, plenty
of haploid maize seeds can be easily obtained for its breeding (Shatskaya et al. 1994; Chang and Coe 2009; Elizabeth
and William 2009; Chaikam 2012). For example, the
frequency of maize haploids induced by MH1 was up to
8.0% (Eder and Chalyk 2002). However, the effectiveness of
corn inducer on the induction of haploid wheat plants has
not been investigated yet. Haploid dwarf male sterile wheat
plants were achieved for the first time in this study by using
DMSW and corn inducer. Comparing with normal inbred
maize line, corn inducer didn’t show any increased induction effect on the formation of haploid wheat embryos
(Table 1). It is inferred that the corn inducer cannot induce
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Figure 5 Fertile traits of the haploid plants derived from the
dwarf male sterile wheat induced by maize pollens. A: The ms2
haploid sterile plants had anthers but no pollens; B: The Ms2 haploid
sterile plants had no anthers.
haploid plants in wheat with a high rate as it does in maize.
In addition, the frequencies for haploid wheat embryos formation and plantlets induction induced by either the corn
inducer or the normal maize inbred line were very low in
this study comparing with other studies by inbred maize
lines or hybrid maize varieties (Cai et al. 2004; Chen et al.
2013), and it might be related to the used DMSW or maize
lines and environmental conditions (Suenaga and Nakajima
Page 6 of 8
1989; Inagaki and Tahir 1990; Wang 1998; Cai et al. 2004;
Chen et al. 2013).
Sun et al. (1999) obtained homozygote of Taigu genic
male sterile wheat plants by chromosome elimination
induced by maize pollens, and identified the plants by
chromosome constitution. They also tried to maintain
the new precious material for long term through cryopreservation by vitrification because of its sterile feature.
We successfully obtained haploid dwarf male sterile wheat
plants by the similar strategy, and identified the plants
combining by chromosome configuration (Figure 2),
cytology (Figure 3), botanical traits (Figure 4, Figure 5),
and molecular markers (Figure 6). It is proved that the
male sterile wheat plants only carrying Ms2 gene can
be easily achieved by the intergeneric cross of wheat
and maize, and there is no need to keep the germplasm
by cryopreservation. Especially, the Ms2 haploid or
homozygous plants can be conveniently recognized by
the short plant height controlled by Rht10 dwarfing
gene (Figure 4), and it can be used as an ideal material
to further clone Ms2 genes in avoid of the interference
of ms2 in the diploid dwarf male sterile wheat. Next, we
plan to obtain doubled haploid Ms2 wheat plants using
this approach, and analyze the plants by transcriptomics
strategies such as RNA-Seq and proteomics techniques
such as mass spectrometry comparing with the doubled
haploid ms2 plants derived from the same DMSW material
correspondingly for the screening of differentially expressed
genes and proteins between the two kinds of plants. And
then, the differentially expressed genes or proteins will be
confirmed by searching the sequenced wheat genomics
and functionally analyzed by genetic transformation for
the possible isolation of candidate Ms2 gene.
Conclusions
Haploid dwarf male sterile male wheat plants were obtained in this study by using corn inducer and normal
inbred maize line. The efficiencies for wheat haploid embryos formation and plantlets production induced by the
two types of corn lines had no significant difference. The
haploid plants were further identified by botanical traits,
Figure 6 Detection of the rescued plants derived from the dwarf male sterile wheat induced by maize pollens and control diploid
plants by molecular market of DF-MR2 for Rht2 gene or Ms2 gene. M: Marker ladder; 1–11: The rescued haploid plants from DMSW. The
plants numbered with 1, 2, 4, 5, 6, 8, 10 and 11 showed the Rht2-specific band, meaning they were the haploid type of Ms2. The plants
numbered with 3, 7, and 9 didn’t have the Rht2-specific band, meaning they were the haploid type of ms2. 12: Chinese Spring, without the
Rht2-specific band; 13: DS987, showing the Rht2-specific band.
Zhang et al. Botanical Studies 2014, 55:26
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cytology, cytogenetics, and molecular marker, and will
be potentially used for the cloning of MS2 dominant
male gene.
Abbreviations
2,4-D: 2,4-dichlorophenoxyacetic acid; CTAB: Cetyltrimethylammonium
bromide; CS: Chinese Spring; DMSW: Dwarf male sterile wheat;
EDTA: Ethylenediaminetetraacetic acid; EMS: Ethyl methane sulfonate;
MS: Murashige and Skoog basal salt mixture; TSD: Tandem segmental
duplication.
Competing interests
The authors declare that they have no competing interests of this research.
Authors’ contributions
Y-XG and L-ZS designed the experiments together. Z-W, W-K and Y-XG made
the intergeneric cross between wheat and maize. Z-W and D-LP determined
the guard cells of the generated plants. X-LL, Z-W, W-K, and Y-XG investigated
the botanic traits of the obtained haploid plants. M-HL and L-ZS finished the
chromosome observation of the rescued and control plants. Z-W and W-K
detected the plants by molecular marker. Y-XG and Z-W analyzed data, wrote
and revised the manuscript. All authors read and approved the final manuscript.
Acknowledgments
We are grateful to Dr. Jiarui Li at Bayer Cropscience, USA, for critical revision
of this manuscript. This research was financially supported in part by grant
from the Ministry of Science and Technology of China (2011BAD35B03).
Received: 20 December 2013 Accepted: 17 February 2014
Published: 20 February 2014
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